Chymotrypsins and subtilisins preferentially hydrolyze peptide bonds in which the carbonyl of the amide link cleaved is donated by N-acyl derivatives of hydrophobic L-amino acid residues. The results of earlier studies by us and others have been used to design new conformationally rigid substrates of these enzymes in order to chemically probe the shape and dimensions of the active sites of the proteins, and to investigate the nature of the interactions between substrate and enzyme. The new substrates are esters of alpha-substituted 1,2,3,4-tetrahydro-3-phenanthroic acid (alpha-substituents:NHAc; H; OAc), various ring-methoxylated 2-acetamido-1,2,3,4-tetrahydro-2-naphthoic acids, and peptide esters in which 2-amino-1,2,3,4-tetrahydro-2-naphthoyl is the C-terminal residue. Whether the locked compounds are good models for the reactive conformations of specific substrates will be ascertained by a variety of structure-reactivity studies. Work completed with the serine proteases suggests the possibility that chymotrypsin- and subtilisin-catalyzed hydrolysis of specific and nonspecific activated ester substrates proceed by different mechanisms; acylation of the enzymes by nonspecific substrates could involve intermediacy of an acylimidazole whereas acylation by specific substrates probably proceeds through an acylserine. The validity of the acylimidazole hypothesis for activated esters of nonspecific substrates will be probed using solvent deuterium isotope effects, the reactivity of anhydrochymotrypsin, and C-13 nmr. The p-nitrophenyl esters of 4-aryl-2-ketobutyric acids are suggested as specific acylating agents of His 57 of alpha-chymotrypsin. The proposed work provides a chemical complement to X-ray crystallographic studies of the structural specificity, stereochemical selectivity, and mechanistic action of the chymotrypsins and subtilisins, which are enzymes of convergent evolution.